Venezuelan Equine Encephalitis virus (VEEV) is a dangerous, NIH/NIAID category B human pathogen and a potential bioterrorism threat. Outbreaks of VEEV occur in Central America and have previously spread into the United States. The potentially devastating effects of the virus reemergence in the U.S. demand an effective vaccine to protect population. Currently, live attenuated TC-83 vaccine is used under IND protocol for vaccination of personnel at risk. The vaccine causes adverse effects, whereas some individuals do not develop neutralizing antibodies. The efforts to develop new VEEV vaccines are underway. However, because vaccine development is a lengthy process and the supply of TC-83 vaccine is limited, the U.S. may soon experience a shortage of VEEV vaccine, which can leave the U.S. population unprotected. We propose a revolutionary new technology for vaccination against VEEV and potentially, other viral diseases. Medigen has recently developed the infectious DNA (i-DNA) vaccination technology, which represents a unique combination of conventional DNA immunization with high efficacy of live attenuated vaccines. The key feature of this technology is that live attenuated virus is launched in vivo from i-DNA plasmid carrying a molecular clone of VEEV vaccine with enhanced safety and immunogenic features. In preliminary studies we have shown that injection in vivo of the prototype i-DNA derived from the TC-83 vaccine has successfully launched live attenuated vaccine in mice. Here we propose the development and feasibility evaluation of novel i-DNA VEEV vaccine for safe and efficient vaccination against VEEV based on the rational design of i-DNA clones and i-DNA immunization technology. In Specific Aim I, we will apply innovative silent mutagenesis method to introduce additional genetic changes into the prototype TC-83 i-DNA in order to secure attenuated phenotype and to generate i-DNA clones with high levels of safety and immunogenicity. The i-DNA clones will be transfected in CHO cells and live attenuated viruses will be harvested from culture media. Safety of i-DNA-derived viruses will be evaluated in mice in collaboration with the Institute of Human Virology, University of Maryland. In Specific Aim II we will vaccinate BALB/c mice with i-DNA constructs in order to induce immune response and to evaluate immunogenicity in vivo. Further, the efficacy of the most promising i-DNA VEEV vaccine will be evaluated in a virulent VEEV challenge experiment at Southwest Foundation for Biomedical Research (SFBR), San Antonio, TX. Finally, reversion studies in vivo will be also conducted at SFBR to demonstrate genetic stability of i-DNA - derived VEEV vaccine. The goal of this 2-year research is the identification of i- DNA VEEV vaccine clone with the optimal safety and immunogenicity profiles for future evaluation and challenge experiments in non-human primates. Our preliminary results suggest that the rational vaccine design and i-DNA technology can provide a revolutionary solution for VEEV vaccine by improving safety, genetic stability, and immunogenicity, and by eliminating many costly steps of the conventional manufacturing process. Essentially, live attenuated vaccine will be manufactured within the immunized individuals. This technology also utilizes many advantages of DNA vaccines (genetic homogeneity and stability, low cost of manufacturing, storage, and transportation, no cold chain) and, more importantly, enhances immunogenicity. Recombinant i-DNA clones produced in bacteria contains CpG motifs that activate TLR9 and MyD88-dependent signaling pathways resulting in robust production of cyto- and chemokines, which induce strong priming effects and stimulate acquired virus- specific immune responses. The final i-DNA VEEV vaccine will represent a novel class of vaccines combining the advantages of DNA and live attenuated vaccines and preserving the backbone of a classic vaccine with a history of human use. The i-DNA technology can be easily adapted for the development of other vaccines including live attenuated vaccines for WEEV, EEEV, other alphaviruses, and flaviviruses. If successful, this technology can potentially transform the field of live attenuated vaccines for many viral diseases.